Neurodegenerative disorders are generally characterized by disease signifying protein deposits. Moreover, in a number of neurodegenerative diseases mutations causing genetically inherited variants of the disease were associated with the genes encoding the protein deposits, their precursors or their modulating enzymes. Functional analysis of these genetic variants fundamentally helped to understand disease associated mechanisms of Alzheimer's disease (AD) and Parkinson's disease (Gasser et al., 2011, Haass et al., 2007). Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are the extreme ends of a spectrum of overlapping neurodegenerative disorders variably associated with dementia, personality changes, language abnormalities and progressive muscle weakness (Josephs et al., 2011; Mackenzie et al., 2010; Rademakers et al., 2012). Research into ALS and FTLD was dramatically accelerated by the identification of the RNA/DNA binding protein TDP-43 (Tar DNA binding protein of 43 kDa) as an abundant deposited protein (Arai et al., 2011; Neumann et al., 2006) and by the discovery that mutations in TARDBP cause familial variants of both diseases (Benajiba et al., 2009; Sreedharan et al., 2008). The majority of cases show intracellular inclusions that are strongly positive for phosphorylated TDP-43. These findings also helped to develop the concept that ALS and FTLD are multisystem disorders with overlapping clinical and pathological characteristics and similar functional and genetic causes (Rademakers et al., 2012; Sieben et al., 2012) and which are therefore classified as FTLD-TDP, FTLD/ALS-TDP or ALS-TDP. Besides TDP-43, and the long known SOD1 (super oxide dismutase 1) gene, a number of other ALS and/or FTLD related genes/risk factors were discovered including FUS (Fused in Sarcoma), OPTN (optineurin), Ataxin 2, Chmp2B, VCP (Valosin containing protein), TMEM106B, GRN (Progranulin), PFN (Profilin) and the C9orf72 gene. Pathological repeat expansions in C9orf72 have been found in about 40% of familial ALS patients and 20% of familial FTLD, demonstrating that C9orf72 is the most common genetic cause for these incurable disorders.
Recently, expansion of a GGGGCC hexanucleotide repeat in the C9orf72 gene has been identified as the most common pathogenic mutation in families with autosomal dominant FTLD, FTLD/ALS and ALS (DeJesus-Hernandez et al. 2011; Renton et al. 2011; Gijselinck et al. 2012). It was further revealed that the hexanucleotide repeat expansion within the regulatory region of the gene C9orf72 is the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. The hexanucleotide repeat expansion is located upstream of the C9orf72 open reading frame, either in the first intron or the promoter region, depending on the transcript isoform (see FIG. 8A). Although the extreme GC-content precludes sequencing in patients, the number of GGGGCC repeat units is believed to be at least several hundred compared to less than 25 in healthy controls (van der Zee et al. 2012). The pathomechanisms leading to disease however remained unclear.
Patients with a C9orf72 repeat expansion have clinical symptoms similar to other FTLD/ALS-TDP patients, but show several unique pathological features (Al-Sarraj et al. 2011; Boxer et al. 2011; Bigio et al. 2012; Whitwell et al. 2012). Aggregates of phosphorylated TDP-43 are accompanied by abundant dot-like and star-shaped phospho-TDP-43-negative neuronal cytoplasmic inclusions in particular in the cerebellum, hippocampus and frontotemporal neocortex that can only be identified with antibodies for p62, ubiquitin or the related ubiquilins. These phospho-TDP-43 negative aggregates are highly characteristic of diseased C9orf72 mutation carriers and are absent in other variants of FTLD/ALS-TDP. The identity of the disease protein(s) in these inclusions and their relation to the C9orf72 hexanucleotide repeat expansion has remained elusive. From research on other neurodegenerative diseases with repeat expansion outside the open reading frame two main pathomechanism have been proposed.
Repeat expansions in non-coding regulatory regions of genes is thought to cause a disease by two different mechanisms that are not mutually exclusive. First, due to the immense length of the repeat expansion transcription and/or splicing may be affected leading to haploinsufficiency (van der Zee et al. 2012). Second, RNA toxicity caused by sequestration of RNA binding proteins may also be causative (Ranum et al., 2006). Currently, evidence for both possibilities exists. The observation of nuclear RNA foci in patients with GGGGCC hexanucleotide repeat expansions, a finding which however is still controversially discussed, suggests that trapping of essential RNA binding proteins may be involved in the disease. Moreover, the finding of decreased expression of C9orf72 mRNA and decreased transcriptional activity of the C9orf72 promoter on intermediate (7-24 repeats) alleles (DeJesus-Hernandez et al. 2011; Gijselinck et al. 2012; van der Zee et al. 2012) implies a loss of function as a disease causing mechanism. These scenarios are not mutually exclusive and may even occur in parallel.
It is unclear how the C9orf72 repeat expansion leads to the characteristic p62-positive/TDP-43-negative inclusions and the subsequent neurodegeneration. There are over 150 papers on C9orf72 genetics and pathology. However, functional data are still very limited.
We disclose herein for the first time that most of these characteristic inclusions contain poly-(Gly-Ala) and to a lesser extent poly-(Gly-Pro) and poly-(Gly-Arg) dipeptide-repeat proteins (DPR) which are generated by non-ATG-initiated translation from the expanded GGGGCC repeats in three reading frames. These findings directly link the FTLD/ALS-associated genetic mutation to the characteristic pathology in patients with C9orf72 hexanucleotide expansion.
So far there is only evidence of a non-ATG-initiated translation of exonic repeat regions in two diseases (Zu et al. 2011). ATXN8 encodes a natural poly-Q stretch that can cause poly-Q inclusions upon repeat expansion in spinocerebellar ataxia type 8 (SCA8) patients. The expanded CAG-repeat is translated in all three reading frames (poly-Q, poly-A and poly-S) even after removal of the endogenous start codon. Poly-Q and poly-A have been found in patient aggregates. Furthermore, myotonic dystrophy type 1 (DM1) is caused by CTG-expansion in the 3′UTR of the gene DMPK. The translation into rare poly-Q aggregates in DM1 patients and mouse models was discovered. The underlying mechanism was named repeat-associated non-ATG-initiated translation (RAN) and is patented for tri-, tetra-, and penta-nucleotide repeat disorders (WO2010/115033 A9). RAN translation has not been shown for intronic repeats or hexanucleotide repeats. In one commentary article Laura Ranum speculates about RAN-translation in C9orf72 patients (Ashizawa and Ranum 2012). It is also possible that translation of DPR proteins is initiated from non-canonical start codon 5′ of the repeat region (Ivanov, I. P. et al. 2011; Peabody D. S. 1989; Touriol C. et al., 2003)
We show for the first time a non-ATG-initiated translation of an intronic repeat expansion, which causes p62-positive/TDP43-negative aggregates of poly-GA, poly-GP and poly-GR. This unusual translation mechanism and the highly abnormal product will facilitate more selective therapeutic approaches than possible for other neurodegenerative diseases. Inhibiting the abnormal dipeptide-repeat protein generation or aggregation will prevent or delay disease progression in mutation carriers. DPR pathology can be used to prevent, delay or cure FTD/ALS in C9orf72 mutation carriers by destabilizing the C9ORF72 (intronic) RNA specifically (e.g., siRNA, anti-sense, alter C9orf72 splicing), inhibiting repeat transcription and/or translation into DPR (screen for RAN-specific translation inhibitors), and preventing DPR aggregation or promoting degradation or clearance (through chemical compounds or immunotherapy).
Similar mechanisms are likely to apply for SCA36, caused by GGCCTG hexanucleotide expansion in NOP56 (Kobayashi et al. 2011). Some of the reading frames encode DPRs as in C9orf72, poly-GP and poly-PR, however with different flanking regions. Therefore, therapeutic drugs discovered for C9orf72 patients are likely to be useful for SCA36 patients too.